Tumour Suppressor Protein

ABSTRACT

We describe a polypeptide which binds and modulates the activity of a tumour suppressor polypeptide, for example p53; a nucleic acid molecule encoding said protein and screening methods which modulate the binding activity of said polypeptide for its target polypeptide(s).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/582,316 which is the U.S. National Stage of InternationalApplication No. PCT/GB2004/003492, filed Aug. 13, 2004 (published inEnglish under PCT Article 21(2)), which in turn claims the benefit ofGreat Britain Application No. 0328690.3, filed Dec. 10, 2003 and U.S.Provisional Application No. 60/554,990, filed Mar. 19, 2004. Thedisclosure of each priority application is incorporated herein byreference in its entirety.

FIELD

The invention relates to a protein that binds and modulates the activityof a tumour suppressor protein, for example p53; a nucleic acid moleculeencoding said protein and screening methods which modulate the bindingactivity of said polypeptide for its target polypeptide.

BACKGROUND

Tumour suppressor genes encode proteins which function to inhibit cellgrowth or division and are therefore important with respect tomaintaining proliferation, growth and differentiation of normal cells.Mutations in tumour suppressor genes result in abnormal cell-cycleprogression whereby the normal cell-cycle check points which arrest thecell-cycle, when, for example, DNA is damaged, are ignored and damagedcells divide uncontrollably. The products of tumour suppressor genesfunction in all parts of the cell (e.g. cell surface, cytoplasm,nucleus) to prevent the passage of damaged cells through the cell-cycle(i.e. G1, S, G2, M and cytokinesis). A number of tumour suppressor geneshave been isolated and sequenced. These include the Retinoblastoma gene(Rb), mutations in which are linked to cancers such as bone(osteocarcoma), bladder, small cell lung and breast cancer, as well asretinoblastoma. The Wilms Tumour 1 gene (WT-1), mutations that arelinked to nephroblastoma and neurofibromatosis.

Arguably the tumour suppressor gene which has been the subject of themost intense research is p53. p53 encodes a protein which functions as atranscription factor and is a key regulator of the cell division cycle.It was discovered in 1978 (Lane and Crawford, 1979) as a protein shownto bind with affinity to the SV40 large T antigen. The p53 gene encodesa 393 amino acid polypeptide with a molecular weight of 53 kDa. One ofthe most important tumour suppression functions of p53 is its ability toinduce apoptosis

Apoptosis, or programmed cell death, is a process by whichmulti-cellular organisms regulate cell number and differentiation. Theprocess is regulated by factors which either induce or preventapoptosis. Inducers of apoptosis include Bcl-2 family members, caspasefamily members and their associated factors Apaf-1 and Fadd. Caspasesare synthesised as proenzymes which become activated after proteolyticcleavage. The active caspase then induces many of the morphological andbiochemical changes associated with apoptosis. Mitochondria play apivotal role in the activation process through the release ofpro-apoptotic factors such as cytochrome c, AIF and Diablo. The releasefrom mitochondria is controlled by the Bcl-2 family of proteins; (e.g.Bcl-2 and Bcl-x1 inhibit release; Bax and Bak induce release).

The polypeptide referred to as iASPP that is described in WO02/12325 isa further example of an agent involved in the regulation of apoptosis.

SUMMARY OF INVENTION

We describe a variant iASPP polypeptide which has characteristics whichare distinct from those described in WO2/12325. The polypeptide,referred to as iASPP6C, is extended at its amino terminus and bindspreferentially to p53 when compared to iASPP. iASPP C6 preferentiallybinds p53 when compared to the shorter version described in W02/12325.The shorter version preferentially binds the apoptosis inducer proteinBcl 2.

iASPP C6 is a ubiquitinated polypeptide which likely controls theturnover of iASPP C6 in vivo. Ubiquitin is a small protein made up of 76amino acids which is highly conserved across species. The most importantfunction assigned to ubiquitin is in regulating protein turnover.Research in recent years has identified many accessory proteins involvedin ubiquitin induced proteolysis. The first step is the ligation ofubiquitin to a target protein which is destined for degradation. This ismediated by three proteins referred to as E1, E2 and E3. Ubiquitin isfirst activated by E1 activating enzyme, a homodimer composed of twoidentical 105 kDa subunits which is ligated to ubquitin via a thioesterbond. Following activation the E1: ubiquitin conjugate is transported byE2 (referred to as a carrier protein). The E2 proteins vary markedly insize but do have some conserved elements. The E2 protein accepts theubiquitin from E1 and forms a second complex again via a thioester bond.The E3 protein may or may not become involved in the final step, whichis the transfer of ubiquitin to a protein substrate. This is followed byrecognition by a protease, which degrades the ubiquitinated protein. Theprotease may be part of a structure referred to as the proteosome whichis a large multi-subunit complex of proteases and associated co-factors.In some examples proteins can become polyubiqitinated, which resultsfrom ubiquitin proteins being ligated to ubiquitin proteins, which arealready ligated to a target protein.

According to an aspect of the invention there is provided an isolatedpolypeptide wherein said polypeptide is represented by the amino acidsequence as shown in FIG. 1 a, or a variant polypeptide which variant ismodified by addition, deletion or substitution of at least one aminoacid residue characterised in that said polypeptide has the followingcharacteristics:

-   -   i) a polypeptide which preferentially binds the tumour        suppressor polypeptide p53 to inhibit the pro-apoptotic activity        of p53 when compared to a polypeptide, or variant thereof, as        represented by the amino acid sequence as shown in FIG. 2 a;    -   ii) a polypeptide which includes at least one amino acid residue        which residue is ubquitinated; and    -   iii) a polypeptide which comprises an amino-terminal polypeptide        domain wherein said domain is represented between amino acid 1        and 483 of the amino acid sequence shown in FIG. 1 a.

In a preferred embodiment of the invention said polypeptidepreferentially binds p53 when compared to a polypeptide represented bythe amino acid sequence shown in FIG. 2 a.

In a further preferred embodiment of the invention said polypeptide ismodified by addition, deletion or substitution of at least one aminoacid residue wherein said modification is between amino acid residues +1and +483 of the amino acid sequence presented in FIG. 1 a.

Assays to determine the binding of polypeptides, which are hereindisclosed, to for example, p53 are known in the art and described in thepresent application.

In a further preferred embodiment of the invention said polypeptidecomprises the amino acid sequence shown in FIG. 1 a. Preferably saidpolypeptide consists of the amino acid sequence shown in FIG. 1 a.

According to an aspect of the invention there is provided an isolatednucleic acid molecule wherein said nucleic acid molecule encodes apolypeptide according to the invention.

In a preferred embodiment of the invention said nucleic acid molecule isrepresented by the nucleic acid sequence shown in FIG. 1 b or a nucleicacid molecule which hybridises to the sequence shown in FIG. 1 b understringent hybridisation conditions and which encodes a polypeptideaccording to the invention.

In a preferred embodiment of the invention said nucleic acid moleculeconsists of the nucleic acid sequence shown in FIG. 1 b.

In a further preferred embodiment of the invention said isolated nucleicacid molecule is a cDNA. In an alternative preferred embodiment of theinvention said nucleic acid molecule is genomic DNA.

According to a further aspect of the invention there is provided avector comprising a nucleic acid molecule according to the invention.Preferably said vector is an expression vector adapted for recombinantexpression of said polypeptide.

Preferably, said vector is adapted for prokaryotic gene expression. Inan alternative embodiment of the invention said vector is adapted foreukaryotic gene expression.

Typically said adaptation includes, by example and not by way oflimitation, the provision of transcription control sequences (promotersequences) which mediate cell/tissue specific expression. These promotersequences may be cell/tissue specific, inducible or constitutive.

Promoter is an art recognised term and includes the following featureswhich are provided by example only, and not by way of limitation.Enhancer elements are cis acting nucleic acid sequences often found 5′to the transcription initiation site of a gene (enhancers can also befound 3′ to a gene sequence or even located in intronic sequences and istherefore position independent). Enhancers function to increase the rateof transcription of the gene to which the enhancer is linked. Enhanceractivity is responsive to trans acting transcription factors(polypeptides) which have been shown to bind specifically to enhancerelements. The binding/activity of transcription factors (please seeEukaryotic Transcription Factors, by David S Latchman, Academic PressLtd, San Diego) is responsive to a number of environmental cues whichinclude, by example and not by way of limitation, intermediarymetabolites or environmental effectors, for example temperature.

Promoter elements also include so-called TATA box and RNA polymeraseinitiation selection (RIS) sequences which function to select a site oftranscription initiation. These sequences also bind polypeptides whichfunction, inter alia, to facilitate transcription initiation selectionby RNA polymerase.

Adaptations also include the provision of selectable markers andautonomous replication sequences which both facilitate the maintenanceof said vector in either the eukaryotic cell or prokaryotic host.Vectors which are maintained autonomously are referred to as episomalvectors. Episomal vectors are desirable since these molecules canincorporate large DNA fragments (30-50 kb DNA). Episomal vectors of thistype are described in WO98/07876.

Adaptations which facilitate the expression of vector encoded genesinclude the provision of transcription termination/polyadenylationsequences. This also includes the provision of internal ribosome entrysites (IRES) which function to maximise expression of vector encodedgenes arranged in bicistronic or multi-cistronic expression cassettes.

These adaptations are well known in the art. There is a significantamount of published literature with respect to expression vectorconstruction and recombinant DNA techniques in general. Please see,Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory, Cold Spring Harbour, N.Y. and referencestherein; Marston, F (1987) DNA Cloning Techniques: A Practical ApproachVol III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

According to a fourth aspect of the invention there is provided a methodfor the production of the polypeptide according to the inventioncomprising:

-   -   i) providing a cell transformed/transfected with a nucleic acid        molecule according to the invention;    -   ii) growing said cell in conditions conducive to the manufacture        of said polypeptide; and    -   iii) purifying said polypeptide from said cell, or its growth        environment.

In a preferred embodiment of the invention said nucleic acid molecule isthe vector according to the invention.

In a preferred method of the invention said vector encodes, and thussaid recombinant polypeptide is provided with, a secretion signal tofacilitate purification of said polypeptide.

According to a further aspect of the invention there is provided anantibody which binds the polypeptide according to the inventioncharacterised in that said antibody binds said polypeptide between aminoacid residues +1 to +483 of the amino acid sequence shown in FIG. 1 a.

Preferably said antibody does not bind said polypeptide represented bythe sequence +484 to +828 of the amino acid sequence shown in FIG. 1 a.

Antibodies, also known as immunoglobulins, are protein molecules whichusually have specificity for foreign molecules (antigens).Immunoglobulins (Ig) are a class of structurally related proteinsconsisting of two pairs of polypeptide chains, one pair of light (L)(low molecular weight) chain (κ or λ), and one pair of heavy (H) chains(γ, α, μ, δ and ε), all four linked together by disulphide bonds. Both Hand L chains have regions that contribute to the binding of antigen andthat are highly variable from one Ig molecule to another. In addition, Hand L chains contain regions that are non-variable or constant.

The L chains consist of two domains. The carboxy-terminal domain isessentially identical among L chains of a given type and is referred toas the “constant” (C) region. The amino terminal domain varies from Lchain to L chain and contributes to the binding site of the antibody.Because of its variability, it is referred to as the “variable” (V)region.

The H chains of Ig molecules are of several classes, α, μ, σ, α, and γ(of which there are several sub-classes). An assembled Ig moleculeconsisting of one or more units of two identical H and L chains, derivesits name from the H chain that it possesses. Thus, there are five Igisotypes: IgA, IgM, IgD, IgE and IgG (with four sub-classes based on thedifferences in the ‘constant’ regions of the H chains, i.e., IgG1, IgG2,IgG3 and IgG4). Further detail regarding antibody structure and theirvarious functions can be found in, Using Antibodies: A laboratorymanual, Cold Spring Harbour Laboratory Press.

In a preferred embodiment of the invention said fragment is a Fabfragment.

In a further preferred embodiment of the invention said antibody isselected from the group consisting of: F(ab′)₂, Fab, Fv and Fdfragments; and antibodies comprising CDR3 regions.

Preferably said fragments are single chain antibody variable regions(scFV's) or domain antibodies. If a hybidoma exists for a specificmonoclonal antibody it is well within the knowledge of the skilledperson to isolate scFv's from mRNA extracted from said hybridoma via RTPCR. Alternatively, phage display screening can be undertaken toidentify clones expressing scFv's. Domain antibodies are the smallestbinding part of an antibody (approximately 13 kDa). Examples of thistechnology is disclosed in U.S. Pat. No. 6,248,516, U.S. Pat. No.6,291,158, U.S. Pat. No. 6,127,197 and EP0368684 which are allincorporated by reference in their entirety.

A modified antibody, or variant antibody and reference antibody, maydiffer in amino acid sequence by one or more substitutions, additions,deletions, truncations which may be present in any combination. Amongpreferred variants are those that vary from a reference polypeptide byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid by another amino acid of likecharacteristics. The following non-limiting list of amino acids areconsidered conservative replacements (similar): a) alanine, serine, andthreonine; b) glutamic acid and asparatic acid; c) asparagine andglutamine d) arginine and lysine; e) isoleucine, leucine, methionine andvaline and f) phenylalanine, tyrosine and tryptophan. Most highlypreferred are variants which show enhanced biological activity.

Preferably said antibody is a humanised or chimeric antibody.

A chimeric antibody is produced by recombinant methods to contain thevariable region of an antibody with an invariant or constant region of ahuman antibody.

A humanised antibody is produced by recombinant methods to combine thecomplementarity determining regions (CDRs) of an antibody with both theconstant (C) regions and the framework regions from the variable (V)regions of a human antibody.

Chimeric antibodies are recombinant antibodies in which all of theV-regions of a mouse or rat antibody are combined with human antibodyC-regions. Humanised antibodies are recombinant hybrid antibodies whichfuse the complimentarity determining regions from a rodent antibodyV-region with the framework regions from the human antibody V-regions.The C-regions from the human antibody are also used. The complimentaritydetermining regions (CDRs) are the regions within the N-terminal domainof both the heavy and light chain of the antibody to where the majorityof the variation of the V-region is restricted. These regions form loopsat the surface of the antibody molecule. These loops provide the bindingsurface between the antibody and antigen.

Antibodies from non-human animals provoke an immune response to theforeign antibody and its removal from the circulation. Both chimeric andhumanised antibodies have reduced antigenicity when injected to a humansubject because there is a reduced amount of rodent (i.e. foreign)antibody within the recombinant hybrid antibody, while the humanantibody regions do not elicit an immune response. This results in aweaker immune response and a decrease in the clearance of the antibody.This is clearly desirable when using therapeutic antibodies in thetreatment of human diseases. Humanised antibodies are designed to haveless “foreign” antibody regions and are therefore thought to be lessimmunogenic than chimeric antibodies.

According to a further aspect of the invention the invention there isprovided a polypeptide according to the invention for use as apharmaceutical.

According to a further aspect of the invention there is provided anucleic acid according to the invention for use as a pharmaceutical.

In a preferred embodiment of the invention said pharmaceutical furthercomprises a diluent, carrier or excipient.

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents, such aschemotherapeutic agents.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. When antibodies are used therapeutically, a preferred routeof administration is by pulmonary aerosol. Techniques for preparingaerosol delivery systems containing antibodies are well known to thoseof skill in the art. Generally, such systems should utilize componentswhich will not significantly impair the biological properties of theantibodies, such as the paratope binding capacity (see, for example,Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences,18th edition, 1990, pp 1694-1712; incorporated by reference). Those ofskill in the art can readily determine the various parameters andconditions for producing antibody aerosols without resort to undueexperimentation. When using antisense preparations of the invention,slow intravenous administration is preferred.

The compositions of the invention are administered in effective amounts.An “effective amount” is that amount of a composition that alone, ortogether with further doses, produces the desired response. In the caseof treating a particular disease, such as cancer, the desired responseis inhibiting the progression of the disease. This may involve onlyslowing the progression of the disease temporarily, although morepreferably, it involves halting the progression of the diseasepermanently. This can be monitored by routine methods or can bemonitored according to diagnostic methods of the invention discussedherein.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. It is generally preferredthat a maximum dose of the individual components or combinations thereofbe used, that is, the highest safe dose according to sound medicaljudgment. It will be understood by those of ordinary skill in the art,however, that a patient may insist upon a lower dose or tolerable dosefor medical reasons, psychological reasons or for virtually any otherreasons.

The pharmaceutical compositions used in the foregoing methods preferablyare sterile and contain an effective amount of for example, a dominantnegative iASPPC6 or nucleic acid encoding a dominant negative iASPPC6,for producing the desired response in a unit of weight or volumesuitable for administration to a patient. The response can, for example,be measured by determining the signal transduction inhibited by thedominant negative iASPP C6, composition via a reporter system, bymeasuring downstream effects such as gene expression, or by measuringthe physiological effects of the iASPPC6 composition, such as regressionof a tumour, decrease of disease symptoms, modulation of apoptosis, etc.

The doses of dominant negative iASPPC6 polypeptide or nucleic acidadministered to a subject can be chosen in accordance with differentparameters, in particular in accordance with the mode of administrationused and the state of the subject. Other factors include the desiredperiod of treatment. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localized delivery route) may beemployed to the extent that patient tolerance permits.

In general, doses of dominant negative iASPPC6 are formulated andadministered in doses between 1 ng and about 500 mg, and between 10 ngand 100 mg, according to any standard procedure in the art. Wherenucleic acids encoding dominant negative iASPPC6 are employed, doses ofbetween 1 ng and 0.1 mg generally will be formulated and administeredaccording to standard procedures. Other protocols for the administrationof iASPPC6 compositions will be known to one of ordinary skill in theart, in which the dose amount, schedule of injections, sites ofinjections, mode of administration (e.g., intra-tumoral) and the likevary from the foregoing. Administration of iASPPC6 compositions tomammals other than humans, e.g. for testing purposes or veterinarytherapeutic purposes, is carried out under substantially the sameconditions as described above. A subject, as used herein, is a mammal,preferably a human, and including a non-human primate, cow, horse, pig,sheep, goat, dog, cat or rodent.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptable compositions. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients. Suchpreparations may routinely contain salts, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

iASPPC6 compositions may be combined, if desired, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation of iASPP C6polypeptides or nucleic acids, which is preferably isotonic with theblood of the recipient. This preparation may be formulated according toknown methods using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation also may be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example, as a solution in 1,3-butane diol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or di-glycerides. In addition, fatty acidssuch as oleic acid may be used in the preparation of injectables.Carrier formulation suitable for oral, subcutaneous, intravenous,intramuscular, etc. administrations can be found in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

In a preferred embodiment of the invention said nucleic acid molecule isan inhibitory RNA (RNAi) molecule or antisense nucleic acid molecule.

In a preferred embodiment of the invention said nucleic acid molecule isselected from the group consisting of an antisense molecule or aninhibitory RNA molecule designed with reference to the nucleic acidsequence shown in FIG. 1 b. Preferably said antisense or inhibitory RNAmolecule is designed to that part of said nucleic acid sequence whichencodes an amino acid sequence as defined by amino acid residues +1 to+483 as shown in FIG. 1 a.

As used herein, the term “antisense molecule” or “antisense” describesan oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridises under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridisation with the target gene or transcript. Those skilled in theart will recognise that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence. It ispreferred that the antisense oligonucleotide be constructed and arrangedso as to bind selectively with the target under physiologicalconditions, i.e., to hybridise substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon the iASPP6C nucleic acid sequences providedherein, or upon allelic or homologous genomic and/or cDNA sequences, oneof skill in the art can easily choose and synthesise any of a number ofappropriate antisense molecules for use in accordance with the presentinvention. For example, a “gene walk” comprising a series ofoligonucleotides of 15-30 nucleotides spanning the length of iASPP6Cnucleic acid can be prepared, followed by testing for inhibition of thecorresponding iASPP6C expression. Optionally, gaps of 5-10 nucleotidescan be left between the oligonucleotides to reduce the number ofoligonucleotides synthesised and tested.

In order to be sufficiently selective and potent for inhibition, suchantisense oligonucleotides should comprise at least 10 and, morepreferably, at least 15 consecutive bases which are complementary to thetarget, although in certain cases modified oligonucleotides as short as7 bases in length have been used successfully as antisenseoligonucleotides (Wagner et al., Nature Biotechnol. 14:840-844, 1996).Most preferably, the antisense oligonucleotides comprise a complementarysequence of 20-30 bases. Although oligonucleotides may be chosen whichare antisense to any region of the gene or mRNA transcripts, inpreferred embodiments the antisense oligonucleotides correspond toN-terminal or 5′ upstream sites such as translation initiation,transcription initiation or promoter sites. In addition, 3′-untranslatedregions may be targeted. Targeting to mRNA splicing sites has also beenused in the art but may be less preferred if alternative mRNA splicingoccurs. In addition, the antisense is targeted, preferably, to sites inwhich mRNA secondary structure is not expected (see, e.g., Sainio etal., Cell Mol. Neurobiol. 14(5):439-457, 1994) and at which proteins arenot expected to bind. Finally, although iASPP 6C cDNA sequences aredisclosed herein, one of ordinary skill in the art may easily derive thegenomic DNA corresponding to the cDNAs. Thus, the present invention alsoprovides for antisense oligonucleotides which are complementary toiASPP6C genomic DNA. Similarly, antisense to allelic or homologous cDNAsand genomic DNAs are enabled without undue experimentation.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognised methods which may be carried out manually or by an automatedsynthesiser. They also may be produced recombinantly by vectors.

In a preferred embodiment of the invention there is provided atranscription cassette comprising a nucleic acid sequence operativelylinked to a promoter which promoter transcribes said nucleic acidmolecule to produce an antisense nucleic acid molecule, said sequenceselected from the group consisting of:

-   -   i) a nucleic acid sequence, or part thereof, as represented in        FIG. 1 b;    -   ii) a nucleic acid sequence which hybridises to the sense        sequence presented in FIG. 1 b and which encodes a polypeptide        according to the invention.

A recent technique to specifically ablate gene function is through theintroduction of double stranded RNA, also referred to as inhibitory RNA(RNAi), into a cell which results in the destruction of mRNAcomplementary to the sequence included in the RNAi molecule. The RNAimolecule comprises two complementary strands of RNA (a sense strand andan antisense strand) annealed to each other to form a double strandedRNA molecule. The RNAi molecule is typically derived from exonic orcoding sequence of the gene which is to be ablated.

Recent studies suggest that RNAi molecules ranging from 100-1000 bpderived from coding sequence are effective inhibitors of geneexpression. Surprisingly, only a few molecules of RNAi are required toblock gene expression which implies the mechanism is catalytic. The siteof action appears to be nuclear as little if any RNAi is detectable inthe cytoplasm of cells indicating that RNAi exerts its effect duringmRNA synthesis or processing.

In a further preferred embodiment of the invention there is provided atranscription cassette comprising a nucleic acid molecule, or partthereof, selected from the group consisting of:

-   -   i) a nucleic acid molecule represented by the nucleic acid        sequence in FIG. 1 b;    -   ii) a nucleic acid molecule which hybridises to the sequence        in (i) above and which encodes a polypeptide according to the        invention; or    -   iii) a nucleic acid molecule which is degenerate because of the        genetic code to the sequences defined in (i) and (ii) above;

wherein said cassette is adapted such that both sense and antisensenucleic acid molecules are transcribed from said cassette.

In a preferred embodiment of the invention said cassette is providedwith at least two promoters adapted to transcribe both sense andantisense strands of said nucleic acid molecule.

In a further preferred embodiment of the invention said cassettecomprises a nucleic acid molecule wherein said molecule comprises afirst part linked to a second part wherein said first and second partsare complementary over at least part of their sequence and furtherwherein transcription of said nucleic acid molecule produces an RNAmolecule which forms a double stranded region by complementary basepairing of said first and second parts.

In a preferred embodiment of the invention said first and second partsare linked by at least one nucleotide base.

In a preferred embodiment of the invention said first and second partsare linked by 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 nucleotide bases.

In a further preferred embodiment of the invention the length of theRNAi molecule is between 100 bp-1000 bp. More preferably still thelength of RNAi is selected from 100 bp; 200 bp; 300 bp; 400 bp; 500 bp;600 bp; 700 bp; 800 bp; 900 bp; or 1000 bp. More preferably still saidRNAi is at least 1000 bp.

In an alternative preferred embodiment of the invention the RNAimolecule is between 15 bp and 25 bp, preferably said molecule is 21 bp.

In a preferred embodiment of the invention said cassette is part of avector.

According to a further aspect of the invention there is provided ascreening method to identify an agent which modulates the interaction ofp53 binding proteins with a p53 polypeptide wherein said methodcomprises the following steps of:

i) forming a preparation comprising a polypeptide according to theinvention and a p53 polypeptide, or sequence variant thereof, and atleast on agent to be tested;

ii) determining the activity of said agent with respect to the bindingof said polypeptide to p53 polypeptide.

According to a further aspect of the invention there is provided ascreening method for the identification of an agent which modulates theinteraction of Bcl-2 binding polypeptides with a Bcl-2 polypeptidewherein said method comprises the steps of:

-   -   i) forming a preparation comprising a polypeptide as represented        by the amino acid sequence shown in FIG. 2 a, or a variant        polypeptide which is modified by addition deletion or        substitution of at least one amino acid residue and a Bcl-2        polypeptide or variant thereof, and at least one agent to be        tested; and    -   ii) determining the activity of said agent with respect to the        binding of said polypeptide for said Bcl-2 polypeptide.

According to a yet further aspect of the invention there is provided ascreening method to identify agents which modulate the ubquitination ofa polypeptide according to the invention comprising the steps of:

-   -   i) forming a preparation comprising a polypeptide according to        the invention, a ubiquitin polypeptide or variant thereof,        polypeptide(s) with the specific activity associated with        ubiquitin conjugating polypeptides and at least one agent to be        tested;    -   ii) determining the activity of said agent with respect to the        conjugation of ubiquitin to said polypeptide.

In a preferred method of the invention said agent is a peptide orpolypeptide.

In a preferred method of the invention said peptide is at least 6 aminoacid residues in length. Preferably the length of saidpeptide/polypeptide is selected from the group consisting of: at least 7amino acid residues; 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20amino acid residues in length. Alternatively the length of saidpeptide/polypeptide is at least 20 amino acid residues; 30; 40; 50; 60;70; 80; 90; or 100 amino acid residues in length.

It will be apparent to one skilled in the art that modification to theamino acid sequence of peptides agents could enhance the binding and/orstability of the peptide with respect to its target sequence. Inaddition, modification of the peptide may also increase the in vivostability of the peptide thereby reducing the effective amount ofpeptide necessary to inhibit p53 binding of iASPP. This wouldadvantageously reduce undesirable side effects which may result in vivo.Modifications include, by example and not by way of limitation,acetylation and amidation. Alternatively or preferably, saidmodification includes the use of modified amino acids in the productionof recombinant or synthetic forms of peptides. It will be apparent toone skilled in the art that modified amino acids include, by way ofexample and not by way of limitation, 4-hydroxyproline, 5-hydroxylysine,N⁶-acetyllysine, N⁶-methyllysine, N⁶,N⁶-dimethyllysine,N⁶,N⁶,N⁶-trimethyllysine, cyclohexyalanine, D-amino acids, ornithine.Other modifications include amino acids with a C₂, C₃ or C₄ alkyl Rgroup optionally substituted by 1, 2 or 3 substituents selected fromhalo (eg F, Br, I), hydroxy or C₁-C₄ alkoxy. Modifications also include,by example and not by way of limitation, acetylation and amidation.

In a preferred embodiment of the invention said peptide sequence isacetylated. Preferably said acetylation is to the amino terminus of saidpeptide.

In a further preferred embodiment of the invention said peptide sequenceis amidated. Preferably said amidation is to the carboxyl-terminus ofsaid peptide.

It will also be apparent to one skilled in the art that peptides couldbe modified by cyclisation. Cyclisation is known in the art, (see Scottet al Chem Biol (2001), 8:801-815; Gellerman et al J. Peptide Res(2001), 57: 277-291; Dutta et al J. Peptide Res (2000), 8: 398-412;Ngoka and Gross J Amer Soc Mass Spec (1999), 10:360-363.

In a further preferred method of the invention said antagonist is anantibody or antibody binding part. Preferably said antibody is amonoclonal antibody or binding part thereof.

In an alternative preferred method of the invention said agent is anaptamer.

Nucleic acids have both linear sequence structure and a threedimensional structure which in part is determined by the linear sequenceand also the environment in which these molecules are located.Conventional therapeutic molecules are small molecules, for example,peptides, polypeptides, or antibodies that bind target molecules toproduce an agonistic or antagonistic effect. It has become apparent thatnucleic acid molecules also have potential with respect to providingagents with the requisite binding properties which may have therapeuticutility. These nucleic acid molecules are typically referred to asaptamers. Aptamers are small, usually stablised, nucleic acid moleculeswhich comprise a binding domain for a target molecule. A screeningmethod to identify aptamers is described in U.S. Pat. No. 5,270,163which is incorporated by reference. Aptamers are typicallyoligonucleotides which may be single stranded oligodeoxynucleotides,oligoribonucleotides, or modified oligodeoxynucleotide oroligoribonucleotides.

The term “modified” encompasses nucleotides with a covalently modifiedbase and/or sugar. For example, modified nucleotides include nucleotideshaving sugars which are covalently attached to low molecular weightorganic groups other than a hydroxyl group at the 3′ position and otherthan a phosphate group at the 5′ position. Thus modified nucleotides mayalso include 2′ substituted sugars such as 2′-O-methyl-; 2-O-alkyl;2-O-allyl; 2′-S-alkyl; 2′-S-allyl; 2′-fluoro-; 2′-halo or2;azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimericsugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanosesugars, and sedoheptulose.

Modified nucleotides are known in the art and include by example and notby way of limitation; alkylated purines and/or pyrimidines; acylatedpurines and/or pyrimidines; or other heterocycles. These classes ofpyrimidines and purines are known in the art and include,pseudoisocytosine; N4,N4-ethanocytosine; 8-hydroxy-N6-methyladenine;4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil;5-bromouracil; 5-carboxymethylaminomethyl-2-thiouracil;5-carboxymethylaminomethyl uracil; dihydrouracil; inosine;N6-isopentyl-adenine; 1-methyladenine; 1-methylpseudouracil;1-methylguanine; 2,2-dimethylguanine; 2-methyladenine; 2-methylguanine;3-methylcyto sine; 5 -methylcyto sine; N6-methyladenine;7-methylguanine; 5-methylaminomethyl uracil; 5-methoxy aminomethyl-2-thiouracil; β-D-mannosylqueosine;5-methoxycarbonylmethyluracil; 5-methoxyuracil; 2methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methyl ester;psueouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil;4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester;uracil 5-oxyacetic acid; queosine; 2-thiocytosine; 5-propyluracil;5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil;5-pentyluracil; 5-pentylcytosine; and 2,6,-diaminopurine;methylpsuedouracil; 1-methylguanine; 1-methylcytosine.

The aptamers of the invention are synthesized using conventionalphosphodiester linked nucleotides and synthesized using standard solidor solution phase synthesis techniques which are known in the art.Linkages between nucleotides may use alternative linking molecules. Forexample, linking groups of the formula P(O)S, (thioate); P(S)S,(dithioate); P(O)NR′2; P(O)R′; P(O)OR6; CO; or CONR′2 wherein R is H (ora salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacentnucleotides through —O— or —S—. The binding of aptamers to a targetpolypeptide is readily tested by assays hereindisclosed.

An embodiment of the invention will now be described by example only andwith reference to the following figures:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is the amino acid sequence of iASPP C6 protein (SEQ ID NO: 1),amino acid sequence underlined is identical to iASPP; FIG. 1 b is thenucleic acid sequence of iASPP6C (SEQ ID NO: 2);

FIG. 2 a is the amino acid sequence of iASPP (SEQ ID NO: 3); FIG. 2 b isthe nucleic acid sequence of iASPP (SEQ ID NO: 4);

FIG. 3 is a sequence alignment of full length iASPP6C (SEQ ID NO: 1 and2) and iASPP (SEQ ID NO: 3 and 4);

FIG. 4A illustrates the expression of iASPP expression in various celllines. Full length iASPP 6C is detected at about 100 kDa. The samplesprobed with antibody LX049.3 are: 2.1 kb iASPP in vitro translated; RKO(colon cancer cell line); HeLa; 293 (kidney); MCF7 (breast); SaOS2(osteosarcoma); HBL100 (breast); H1299 (lung); U937 (lung); U2OS(osteosarcoma); FIGS. 4B, 4C and 4D illustrates that iASPP6C can bedetected by distinct antibodies raised against sequences within bothiASPP and iASPP6C. 4B is a diagram illustrating the relative positionsof the antigens used to generate antibodies LX049.3, SA4.1 and pAb18; 4Cshows that both iASPP6C and iASPP cDNAs were translated in vitro usingunlabelled amino acids. A control reaction was run alongside whichcontained empty vector. The band observed between the two iASPP proteinsin the blot probed with LX049.3 is non-specific. 4D shows the expressionlevels of iASPP6C and iASPP were detected in cell lines using LX049.3.In vitro translated products of the iASPP and iASPP6C cDNAs (IVT) areloaded as positive controls. The positions of the molecular weightmarkers are shown on the right. Anti PCNA antibody PC-10 was used as aloading control for the cell lysates;

FIG. 5 illustrates an immunoprecipitation/western blot using twodifferent iASPP antibodies: LX049.3 is a mouse monoclonal while pAb18 isa rabbit antibody (epitopes are given in the peptide alignment, see FIG.3);

FIGS. 6 a and 6 b illustrates that iASPP 6C is ubiquitinated. Thisprocess results in the generation of the 83 kDa fragment, which isabolished in the presence of MG132 (ubiquitin-proteosome inhibitor).Ubiquitination also appears to be dependent on cell density. The cellswere split according the cell density required and MG132 was added thenext day (16-24 hrs later);

FIG. 7 illustrates that p53 preferentially binds to full length iASPP6C,while Bcl-2 preferentially binds iASPP;

FIG. 8 illustrates the activity of the full length iASPP6C in cells andthat iASPP and p53 are involved in the activation of apoptotic genes butnot cell cycle regulatory genes, and that it also interacts with p63 andp73;

FIG. 9 illustrates that the full length iASPP6C is preferentiallyexpressed in cells rather than iASPP but at different expression levels;

The N-terminus of iASPP6C causes its localisation to the cytoplasm: (a)LX049.3 was used to detect iASPP6C in Saos-2 and H1299 cells. Eithertransfected or endogenous iASPP6C was analysed in Saos-2 cells,alongside endogenous iASPP6C in H1299 cells. (b) V5 epitope-taggedconstructs encoding the regions of iASPP were transfected into Saos-2cells and their subcellular localisation determined byimmunofluorescence using anti-V5 antibody.

The C-terminus of iASPP6C is required for the inhibition of p53. (a)Saos-2 cells were transfected with p53 and the indicated iASPPtruncations, and apoptosis was detected by FACS. (b) Saos-2 cells weretransfected with 1 μg of a luciferase reporter plasmid containing thePIG-3 promoter alongside 50 ng of p53 and 0.25 μg of the iASPP6Cplasmids.

DETAILED DESCRIPTION

Materials and Methods

Cell Culture and Reagents

Cells were grown in culture in Dulbecco's modified Eagle medium(Invitrogen) supplemented with 10% foetal calf serum. The cells used inthis study were Tera (testicular tumour cell line), RKO (coloncarcinoma), Saos-2 (osteosarcoma), H1299 (lung carcinoma), 293(embryonic kidney), SK-MEL-37 (melanoma), MCF7 (mammary epithelial) andU2OS (osteosarcoma). Anti-V5 antibody was purchased from Invitrogen.N-20 CD20Leu FITC-conjugated monoclonal antibody was from BectonDickinson. Transfections throughout were performed by calcium phosphateprecipitation.

Plasmids

The EST containing the cDNA encoding iASPP6C (I.M.A.G.E. clone 4994121)was obtained from MRC Geneservice (Cambridge, U.K.). The cDNA wassubcloned into pcDNA3.1/V5-His-TOPO (Invitrogen). pcDNA3.1 iASPP,pcDNA3.1 ASPP2, pcDNA3.1 Ce-iASPP and pcDNA3 p53 have been describedpreviously (Bergamaschi et al., 2003; Samuels-Lev et al., 2001). TheiASPP6C truncations used in FIG. 10 were generated by PCR-directedcloning into pcDNA3.1/V5-His-TOPO. A modified pcDNA3 vector that has hadtwo V5 sequences inserted 5′ of the polylinker was used to generateN-terminally V5-tagged iASPP6C.

Generation of Anti-iASPP Antibodies

Anti-iASPP6C antibodies pAb18 (rabbit polyclonal) and SA4.1 (mousemonoclonal) were raised against the peptide RLQPALPPEAQSVPELEE (aminoacids 492 to 509 of iASPP6C). Anti iASPP6C mouse monoclonal antibodyLX049.3 was raised against a C-terminal His-tagged fusion proteincontaining amino acids 459 to 639 of iASPP6C. The corresponding cDNA wasamplified by PCR and subcloned into pCRT7/CTTOPO (Invitrogen). Therecombinant iASPP6C fragment was generated in BL21 Star E. coli(Invitrogen) by incubation with 1 mM IPTG for 4 h followed bypurification under denaturing conditions.

Electrophoresis and Immunoblotting

Cells were washed twice in PBS, then scraped into 1 ml PBS and pelletedat 400 g. The cells were lysed by incubating for 30 minutes at roomtemperature in 8M urea, 1M thiourea, 0.5% CHAPS, 50 mM DTT and 24 mMspermine, followed by centrifugation at 20 000 g for 20 minutes at 16°C. 30 μg protein was used for analysis by SDS-PAGE and immunoblotting asdescribed previously (Yap et al., 2000).

Immunoprecipitation

Cells were lysed by incubating on ice in NP40 lysis buffer (50 mM TrispH8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40 and protease inhibitors (completeprotease inhibitor cocktail, Roche)) for 45 minutes followed bycentrifugation for 20 minutes at 20 00 g at 4° C. Between 0.5 and 2 mglysate was precleared by rotating for 1 h at 4° C. with protein Gsepharose beads (Amersham Biosciences). Following removal of the beads,the lysate was transferred to a fresh tube and rotated overnight withblocked protein G sepharose beads at 4° C. and approximately 1 μg ofeither a specific antibody or non-specific mouse or rabbit IgG (Sigma)as controls. The beads were then washed three times in ice cold NP40lysis buffer and the resulting complexes analysed by SDS-PAGE andimmunoblot.

Construction and Transfection of iASPP6C siRNA

Oligonucleotides containing 19 bases of sequence present in both iASPP6Cand iASPP cDNAs were ligated into the pSuper expression plasmid asdescribed previously (Brummelkamp et al., 2002). The plasmids wereverified by sequencing. The complete sequences of the oligonucleotidesused to generate the siRNA are as follows with the cDNA sequences shownin upper case:

sense, 5′gatccccTGTCAACTCCCCCGACAGCttcaagagaGCTGTCGGGGGAGTTGACAtttttggaaa 3′; antisense,5′agcttttccaaaaaTGTCAACTCCCCCGACAGCtctcttgaaGCTGTC GGGGGAGTTGACAggg 3′.

For transfection, 1×10⁶ H1299 cells were plated into 10 cm dishes. Cellswere

transfected with 3 μg of pMACS H-2K^(K) alongside either pSuper orpSuper-si-RNA iASPP (10 μg). 48 h after transfection, cells expressingthe pMACS H-2K^(K) plasmid were separated using the MACS system(Miltenyi Biotec) according to the manufacturer's instructions. Thisgave rise to two populations of cells: H-2K^(K) expressing (transfected)cells and non-expressing (non-transfected cells). Both cell populationswere lysed with RIPA buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris pH8,0.5% deoxycholate, 1% NP40, 0.1 % SDS) on ice for 30 minutes followed bycentrifugation at 20 000 g for 30 minutes at 4° C.

In Vitro Translation and In Vitro Immunoprecipitation

p53 and iASPP6C were translated in vitro with 35S-methionine using theTNT T7 Quick coupled Transcription/Translation System (Promega). Thereticulocyte lysates containing each protein were combined as indicatedand incubated together for 1 h at 30° C. LX049.3 antibody immobilised onprotein G sepharose beads was added to the binding reactions and rotatedat 4° C. for 16 h. The beads were then washed with PBS. The boundproteins were released in SDS sample buffer and analysed by 10%SDS-PAGE. Results were visualised by autoradiography.

Transactivation

The transcriptional assay was carried out as described previously(Samuels-Lev et al., 2001).

Flow Cytometry

Flow cytometry 1×10⁶ Saos-2 cells were plated in 10 cm dishes 24-48 hprior to transfection. All cells were transfected with 2 μg of pCMV CD20as a transfection marker. The following plasmids were transfected asappropriate at the stated amounts: pcDNA3 p53 (1 μg), pcDNA3.1 Ce-iASPP(7.5 μg), pcDNA3.1 iASPP (7.5 μg), pcDNA3.1 iASPP6C(1 μg), pcDNA3.1ASPP2 (10 μg). 2 μg iASPP6C truncations were used in FIG. 11. EmptypcDNA3 vector was used to equalise the total amount of DNA in allsamples. 36 h after transfection, both attached and floating cells wereharvested and analysed as described previously (Hsieh et al., 1997).

Immunofluorescence

Saos-2. cells were seeded on cover slips in 24 well plates at 50%density and transfected with 0.5-3 μg of plasmid encoding the iASPP6Ctruncations. 24 h after transfection the cells were fixed with 200 μl of4% paraformaldehyde in PBS for 12 minutes then permeabilised with 0.1%Triton-X100 in PBS for 4 minutes. Expression of the iASPP6C constructswas detected using anti-V5 antibody (1:100 dilution in 0.2% fish skingelatin) for 40 minutes followed by a TRITC or FITC-conjugated secondaryantibody for 20 minutes.

REFERENCES

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1-4. (canceled)
 5. The isolated nucleic acid molecule according to claim6 wherein said polypeptide consists of the amino acid sequence set forthin SEQ ID NO:
 1. 6. An isolated nucleic acid molecule comprising anucleotide sequence that encodes a polypeptide comprising the amino acidsequence set forth in SEQ ID NO:
 1. 7. The isolated nucleic acidmolecule according to claim 6, wherein said nucleic acid moleculecomprises the nucleic acid sequence set forth in SEQ ID NO: 2 or anucleic acid molecule which hybridises to the sequence set forth in SEQID NO: 2 under stringent hybridisation conditions, wherein said nucleicacid sequence encodes the polypeptide comprising the amino acid sequenceset forth in SEQ ID NO:
 1. 8. The isolated nucleic acid moleculeaccording to claim 6 wherein said nucleic acid molecule consists of thenucleic acid sequence set forth in SEQ ID NO:
 2. 9. The isolated nucleicacid molecule according to claim 6 wherein said molecule is a cDNA. 10.The isolated nucleic acid molecule according to claim 6 wherein saidmolecule is genomic DNA.
 11. A vector comprising the nucleic acidmolecule according to claim
 6. 12. A method for producing a polypeptide,comprising transforming or transfecting a cell with the vector accordingto claims 11; growing said cell in conditions conducive to themanufacture of said polypeptide; and purifying said polypeptide fromsaid cell, or its growth environment. 13-18. (canceled)
 19. Acomposition comprising the nucleic acid molecule according to claim 6and a pharmaceutically-acceptable carrier.
 20. The composition accordingto claim 6, wherein said nucleic acid molecule is an inhibitory RNAmolecule.
 21. The composition according to claim 6, wherein said nucleicacid molecule is an antisense nucleic acid molecule.
 22. The compositionaccording to claim 20, wherein said inhibitory RNA molecule is designedto that part of said nucleic acid sequence which encodes amino acidresidue 1 to 483 set forth in SEQ ID NO:
 1. 23. The compositionaccording to claim 22, said nucleic acid molecule is provided as atranscription cassette comprising a nucleic acid sequence operativelylinked to a promoter which promoter transcribes said nucleic acidmolecule to produce an antisense nucleic acid molecule, said sequenceselected from the group consisting of: i) a nucleic acid sequence, orpart thereof, set forth in SEQ ID NO: 2; ii) a nucleic acid sequencewhich hybridises to the sense sequence set forth in SEQ ID NO: 2 FIG. 1b—and which encodes the polypeptide set forth in SEQ ID NO:
 1. 24.(canceled)
 25. The composition according to claim 22 wherein saidcassette is provided with at least two promoters adapted to transcribeboth sense and antisense strands of said nucleic acid molecule.
 26. Thecomposition according to claim 22 wherein said cassette comprises anucleic acid molecule wherein said molecule comprises a first partlinked to a second part wherein said first and second parts arecomplementary over at least part of their sequence and further whereintranscription of said nucleic acid molecule produces an RNA moleculewhich forms a double stranded region by complementary base pairing ofsaid first and second parts.
 27. The composition according to claim 26wherein said first and second parts are linked by at least onenucleotide base.
 28. The composition according to claim 23 wherein saidcassette is part of a vector. 29-34. (canceled)
 35. The compositionaccording to claim 21, wherein said antisense is designed to that partof said nucleic acid sequence which encodes amino acid residue 1 to 483set forth in SEQ ID NO:
 1. 36. The isolated nucleic acid moleculeaccording to claim 6, comprising a nucleotide sequence that encodesamino acids 1-483 of SEQ ID NO:
 1. 37. The isolated nucleic acidmolecule according to claim 6, wherein at least one residue of theencoded polypeptide is ubuiqunated.
 38. The isolated nucleic acidmolecule according to claim 6, wherein the encoded polypeptide isacetylated.
 39. The isolated nucleic acid molecule according to claim38, wherein the amino-terminus of the encoded polypeptide is acetylated.40. The isolated nucleic acid molecule according to claim 6, wherein theencoded polypeptide is amidated.
 41. The isolated nucleic acid moleculeaccording to claim 40, wherein the carboxyl-terminus of the encodedpolypeptide is amidated.